JP2009525867A - Methods for inducing substance nucleation - Google Patents

Abstract

A method for inducing nucleation of a material is provided. The disclosed method includes moving the material near or below the phase transition temperature and changing the pressure to induce nucleation of the material. The disclosed method is useful in lyophilization processes, particularly in the lyophilization process of drugs.

Description

  The present invention relates to a nucleation method, and more particularly to a method for inducing nucleation of a phase transition in a material. In this method, the material is first brought to a temperature near or below the phase transition temperature and subsequently depressurized to induce nucleation of the material.

  This application claims priority based on US Provisional Application No. 60 / 771,868, filed February 10, 2006.

  In the freezing phase of the lyophilization process, generally control the random nucleation process to reduce the processing time required to complete the lyophilization and, in the finished product, the product between the vials Both will be highly desirable in the art. In a typical lyophilization process of a drug, a plurality of vials containing a customary aqueous solution are placed on a shelf and they are cooled to a low temperature, generally at a controlled rate. The aqueous solution in each vial is cooled below the thermodynamic freezing temperature of the solution and left in a supercooled metastable liquid state until nucleation occurs.

  The range of nucleation temperatures through the vial can be left randomly between a temperature near the thermodynamic freezing temperature and some value clearly below that thermodynamic freezing temperature (eg, up to about 30 ° C.). Is distributed. This distribution of nucleation temperatures causes the ice crystal structure and, ultimately, the physical properties of the lyophilized product to vary between vials. Furthermore, in order to adjust the size and structure range of ice crystals produced by the natural stochastic nucleation phenomenon, the drying stage of the lyophilization process needs to be very long.

  Additives have been used to increase the nucleation temperature of the supercooled solution. These additives can take many forms. It is well known that certain bacteria (eg, Pseudomonas syringae) synthesize proteins that help nucleate ice formation in supercooled aqueous solutions. Either this bacterium or a protein isolated from them can be added to the solution to increase the nucleation temperature. Several inorganic additives have also demonstrated a nucleation effect. The most common such additive is silver iodide, AgI. In general, any additive or contaminant has the potential to act as a nucleating agent. Freeze-drying vials prepared in an environment containing high levels of particles generally nucleate and freeze at a lower degree of supercooling than vials prepared in a low level particle environment Will.

  All the above nucleating agents are named “additives” because they change the composition of the medium and become the core of the phase transition. These additives are not generally accepted for lyophilized drug products regulated and approved by the US Food and Drug Administration. These additives also do not provide control over time and temperature as the vial nucleates and freezes. Rather, the additive only functions to increase the average nucleation temperature of the multiple vials.

  The ice crystals can themselves act as a nucleating agent for ice formation in supercooled aqueous solutions. In the “ice mist” method, a wet lyophilizer is filled with a cold gas to produce a vapor suspension of small ice particles. These ice particles are carried into the vial and initiate nucleation when they come into contact with the fluid interface.

  The “ice fog” method does not simultaneously control nucleation of multiple vials at a controlled time and temperature. In other words, when cold steam is introduced into the lyophilizer, nucleation events do not occur simultaneously or immediately within all vials. It will take some time for the ice crystals to struggle to reach each vial and begin nucleation, and will be carried against the vials at different locations inside the lyophilizer. The time spent is likely to be different. For large industrial freeze dryers, implementation of the “ice fog” method may require internal convection devices to support a more uniform distribution of “ice fog” throughout the freeze dryer System design changes will be necessary. When cooling the freeze dryer shelf continuously, the time difference between the first vial freezing and the last vial freezing is probably the temperature difference between the vials. This will likely increase the heterogeneity between the vials, possibly in the lyophilized product.

  Pretreatment of vials by scoring, scratching, or roughing has also been used to reduce the degree of supercooling required for nucleation. As with other prior art methods, vial pretreatment does not give any control over time and temperature as individual vials nucleate and freeze, instead, all It only increases the average nucleation temperature of the vial.

  Vibrations have also been used to form phase transition nuclei in metastable materials. Sufficient vibrations to induce nucleation occur at frequencies above 10 kHz and can be created using various devices. This frequency range vibration is often referred to as “ultrasound”, even though frequencies in the range of 10 kHz to 20 kHz are typically within the human audible range. Ultrasonic vibrations often create cavitation, i.e., form small bubbles, in the supercooled solution. In transient or inertial cavitation situations, the bubbles grow rapidly and rupture, causing very high local pressure and temperature fluctuations. The ability of ultrasonic vibrations to induce nucleation in metastable materials is often attributed to disturbances caused by transient cavitation. Other cavitation situations, referred to as stable or non-inertial, are characterized by bubbles that exhibit a stable volume or shape of vibration without failure. US Patent Publication 200320031577 A1 discloses that ultrasonic vibration can induce nucleation even in stable cavitation situations, but no explanation for this phenomenon is presented. UK patent application 2400901A also uses vibrations with a frequency higher than 10 kHz, and the prospect of causing cavitation in the solution, and hence nucleation, is to reduce the atmospheric pressure around the solution or to reduce the volatility in the solution. It is disclosed that it can be increased by dissolving the fluid.

  Electrofreezing has also been used in the past to induce nucleation in supercooled liquids. Electrofreezing is generally achieved by applying a relatively high electric field (approximately 1 V / nm) continuously or in a pulsed manner between closely spaced electrodes immersed in a supercooled liquid or solution. Is done. In typical lyophilization applications, the difficulties associated with electrofreezing methods include relative complexity and cost to implement and maintain, especially for lyophilization applications that use multiple vials or containers. Also, electrofreezing cannot be applied directly to solutions containing ionic species (eg, NaCl).

  In recent years, there have been studies examining the concept of “vacuum-induced surface freezing” (see, eg, US Pat. No. 6,684,524). In such “vacuum-induced surface freezing”, a vial containing an aqueous solution is placed on a temperature controlled shelf in a freeze dryer and initially held at about 10 ° C. The lyophilization chamber is then evacuated to near vacuum pressure (eg 1 mbar). This results in freezing of the surface of the aqueous solution to a depth of a few mm. Subsequently, the vacuum is released and the shelf temperature is lowered below the freezing point of the solution, causing ice crystals to grow from the pre-frozen surface layer through the remainder of the solution. A major challenge to the implementation of this “vacuum-induced surface freezing” method in typical lyophilization applications is the high risk of severe boiling or outgassing of the solution under the conditions presented.

  The improved control of the nucleation process is such that freezing of all unfrozen drug solution vials in the lyophilizer occurs within a narrower temperature and time range, thereby providing greater uniformity between the vials, Making it possible to produce freeze-dried products. Controlling the minimum nucleation temperature affects the crystal structure of the ice formed in the vial and can allow a significantly accelerated lyophilization process.

  Therefore, freezing of the freeze-drying method both reduces the processing time required to complete the freeze-drying and improves the heterogeneity between the product vials in the finished product. There is a need to control the delegation nucleation process in various freezing processes including stages. Therefore, it would be desirable to provide a method having some, or preferably all, of the above characteristics.

  The present invention may be positioned as a method for inducing nucleation of phase transitions in matter. The method includes the steps of bringing the material near or below the phase transition temperature of the material and reducing the pressure to induce nucleation of the phase transition in the material. Including.

  The invention also includes cooling the solution at a predetermined cooling rate; rapidly reducing the pressure to induce nucleation of the solution; and continuing to cool the nucleated solution to a predetermined final temperature. Then, it may be positioned as a method for controlling the freezing process of the solution including the step of freezing the solution. Depressurization is initiated when the solution reaches the desired nucleation temperature or at a desired time after the start of the cooling phase.

  The invention further includes cooling the material near or below the phase transition temperature; rapidly reducing the pressure in the vicinity of the material to induce nucleation of the material; and a predetermined final temperature. Until then, cooling of the nucleated material may be continued to promote solidification of the material, and may be positioned as a solidification method.

  Finally, the present invention provides a method of cooling a gas near or below the phase transition temperature; rapidly reducing pressure to induce nucleation in the gas; and The cooling of the nucleated gas is continued until the final temperature, and the step of condensing the gas may be positioned as a method for controlling the gas condensation process.

  The above and other aspects, features and advantages of the present invention will become more apparent from the following more detailed description, which is illustrated using the following drawings.

  Nucleation is the beginning of a phase transition in a small region of matter. For example, the phase transition can be the formation of crystals from a liquid. The crystallization process (ie, the formation of solid crystals from the solution) may involve freezing of the solution beginning with a nucleation event followed by crystal growth.

  In the crystallization process, nucleation is the stage at which selected molecules dispersed in a solution or other material begin to assemble, creating nanometer-scale clusters and becoming stable at current operating conditions. These stable clusters constitute the nucleus. These clusters need to reach a critical size in order to become stable nuclei. Such critical sizes are usually defined by operating conditions such as temperature, contaminants, supersaturation, and can vary from solution sample to solution sample. During the nucleation event, the atoms in the solution are arranged in a defined periodic manner, which defines the crystal structure.

  Crystal growth is growth following a nucleus that has successfully achieved a critical cluster size. Depending on the conditions, either nucleation or crystal growth can be dominant over the other, resulting in crystals of different sizes and shapes. Control of crystal size and shape constitutes one of the main challenges in industrial manufacturing for drugs and the like.

  The method relates to a method for controlling time and / or temperature when a nucleated phase transition occurs in a material. In freezing applications, the possibility of a substance spontaneously nucleating and initiating a phase change is the degree of subcooling of the substance and the contaminants, additives, structures, or disturbances that provide sites or surfaces for nucleation Related to the presence or absence of

  While the freezing or solidification stage is particularly important in the lyophilization process, existing techniques have resulted in nucleation temperature differences across many vials or containers. Differences in nucleation temperatures tend to produce heterogeneous products and excessively long drying times. On the other hand, the present method produces a product with a higher degree of method control and a more uniform structure and properties in a batch-type solidification method (for example, lyophilization). Unlike some of the prior art techniques for inducing nucleation, the method requires minimal equipment and operational changes to perform.

  In principle, the method can be applied to any processing stage of matter with a nucleation phase transition. Examples of such processing steps are liquid freezing, ice crystallization from aqueous solution, polymer and metal crystallization from melt, inorganic material crystallization from supersaturated solution, protein crystallization, artificial snow Includes liquefaction by production, ice deposition from steam, food freezing, freeze concentration, partial crystallization, cryopreservation, or steam condensation. From a conceptual point of view, the method may be applicable to phase transitions such as melting and boiling.

  The methods disclosed herein represent an improvement over current drug lyophilization methods. For example, inside a large industrial lyophilizer, there can be over 100,000 vials containing products of drugs that need to be frozen and dried. Today's practice in the industry is to cool the solution to a very high degree to ensure freezing of the solution in all vials or containers in the lyophilizer. However, the contents of each vial or container freezes randomly over a temperature range below the freezing point because the nucleation process is not controlled.

  Turning now to the drawing, particularly FIG. 1, a temperature vs. time curve is drawn for six vials of an aqueous solution undergoing a conventional stochastic nucleation process. The figure shows typical nucleation temperature ranges (11, 12, 13, 14, 15 and 16) for solutions in vials. As can be seen, the contents of the vial have a thermodynamic freezing temperature of about 0 ° C., but the solution in each vial is naturally about −− as highlighted by region 18. Nucleates over a wide temperature range from 7 ° C to higher than -20 ° C. Curve 19 shows the temperature of the shelf inside the freeze-drying chamber.

  In contrast, FIGS. 2 and 3 depict temperature versus time curves for solutions undergoing a freezing process with reduced nucleation consistent with the present method. In particular, FIG. 2 shows the temperature vs. time curve (21, 6) of six vials of an aqueous solution undergoing an equilibration cooling process with nucleation induced via chamber vacuum (see Example 2). 22, 23, 24, 25 and 26). The contents of the vials have a thermodynamic freezing temperature of about 0 ° C., but the solution in each vial is still at the same time during decompression and within a very narrow temperature range (as seen in region 28) ( That is, nuclei are formed at −4 ° C. to −5 ° C.). Curve 29 shows the temperature of the shelf inside the lyophilization chamber and also depicts an equilibrated freezing process in which the shelf temperature is kept more or less stable prior to decompression.

  Similarly, FIG. 3 shows the temperature vs. time curve (31 of three vials of an aqueous solution undergoing a dynamic cooling process (see Example 7) with nucleation induced via chamber vacuum. , 32 and 33). Again, the contents of the vial have a thermodynamic freezing temperature of about 0 ° C., but the solution in each vial is nevertheless from about −7 ° C.- Nuclei form in the temperature range of 10 ° C. Curve 39 shows the temperature of the shelf inside the lyophilization chamber and generally depicts a dynamic cooling process in which the temperature of the shelf is actively reduced during or prior to depressurization.

  As illustrated in these figures, the method causes freezing of the drug solution in the lyophilizer within a narrower temperature range (eg, about 0 ° C. to −10 ° C.) and / or simultaneously. This provides improved control of the nucleation process, thereby yielding a lyophilized product with greater homogeneity between vials. Although not demonstrated, it is foreseen that the range of nucleation temperatures induced can still be slightly expanded beyond the phase transition temperature and can extend to about 40 ° C of subcooling. it can.

  Another advantage with this method is that it affects the crystal structure of ice formed inside a frozen vial or container by controlling the lowest nucleation temperature and / or the exact time of nucleation. Be able to. The crystal structure of ice is a variable that affects the time it takes for ice to sublime. Thus, by controlling the ice crystal structure, the overall freeze-drying process can be significantly accelerated.

  In a broad sense, the method for inducing nucleation of a phase transition in a material disclosed herein includes the steps of (i) cooling the material to a temperature near or below the phase transition temperature of the material. And (ii) rapidly reducing the pressure to induce nucleation of the material. Each of these important steps is discussed in more detail below.

Stage 1-Cooling of Substances Illustrative substances useful in the present method include pure substances, gases, suspensions, gels, liquids, solutions, mixtures, or components in solutions or mixtures. Suitable materials for use in the method may include, for example, drug substances, biopharmaceutical substances, foods, chemicals, and products such as wound treatment products, cosmetics, veterinary products, and in-vivo / in-vitro diagnostic related products. May be included. If the substance is a liquid, it may be desirable to dissolve the gas in the liquid. Liquids in a controlled gaseous environment will generally dissolve the gas in them.

  Other illustrative materials useful in the method include biological or biopharmaceutical materials such as tissues, organs, and multicellular structures. For certain biological or pharmaceutical applications, the substance can be a live or attenuated virus; a nucleic acid; a monoclonal antibody; a polyclonal antibody; a biomolecule; a non-peptide analog; a polypeptide, a peptidomimetic and a modified peptide A solution or mixture comprising a protein, including fusion and modified proteins; RNA, DNA and their subclasses; oligonucleotides; viral particles; and analogs of such substances or components thereof. It's okay.

  Drug or biopharmaceutical solutions placed in lyophilized vials or containers would be a good example of a substance that would benefit from the present method. The solution is mostly water and is substantially incompressible. Such drug or biopharmaceutical solutions are also highly pure and generally free of particles that may form sites of nucleation. Uniform nucleation temperature is important to create a consistent and uniform ice crystal structure between vials or containers. The crystal structure of the growing ice also has a significant effect on the time required for drying.

  When applied to the lyophilization process, the material is preferably placed in a chamber, such as a lyophilization chamber. Preferably, the chamber is configured to allow control of the temperature, pressure, and gas atmosphere in the chamber. The gas atmosphere is not limited to these, but argon, nitrogen, helium, air, water vapor, oxygen, carbon dioxide, carbon monoxide, nitrous oxide, nitric oxide, neon, xenon, krypton, methane, hydrogen, propane, Butane and the like, and acceptable mixtures thereof may be included. A preferred gas atmosphere includes an inert gas such as argon having a pressure between about 7 and about 50 psig or more. The temperature in the lyophilizer room is often defined by the lyophilization process, and the shelves in the room are cooled or heated to drive the temperature of the vials or containers and the substance in each vial or container. It is easily controlled through the use of heat transport fluid.

  In accordance with the method, the material is cooled to a temperature near or below its phase transition temperature. In the case of an aqueous solution that has undergone a lyophilization process, the phase transition temperature is the thermodynamic freezing point of the solution. When a solution reaches a temperature below the thermodynamic freezing point of the solution, it is said to be supercooled. When applied to the freezing process of an aqueous solution, the present method can be used when the extent of the supercooling range is in the vicinity of or below the phase transition temperature to about 40 ° C. of supercooling, more preferably about It is effective when it is between 3 ° C and 10 ° C of supercooling. In some of the examples described below, the method of inducing nucleation works well even when the solution has only been subcooled to about 1 ° C. below its thermodynamic freezing point.

  When a material is at a temperature below its phase transition temperature, it is often referred to as being in a metastable state. A metastable state is a chemical or biological state that is unstable and transient, but has a relatively long lifetime. A metastable material is temporarily present in a phase or state that is not its equilibrium phase or state. If there is no change in the material or its environment, the metastable material ultimately transitions from its non-equilibrium state to its equilibrium state. Examples of metastable materials include supersaturated solutions and supercooled liquids.

  A typical example of a metastable material would be liquid water at atmospheric pressure and a temperature of -10 ° C. Since the normal freezing point is 0 ° C., liquid water does not exist thermodynamically at this temperature and pressure, but it can exist without a nucleation event or structure that initiates the ice crystallization process. Extremely pure water can be cooled to very low temperatures (−30 ° C. to −40 ° C.) at atmospheric pressure and still remain liquid. Such supercooled water is in a non-equilibrium, thermodynamically metastable state. It lacks only the nucleation events that cause the phase transition to start and thereby return to equilibrium.

  As discussed above, this method of inducing nucleation of phase transitions within a material, or a method of freezing a material, with various cooling profiles, including, for example, an equilibrated cooling environment or a dynamic cooling environment. Available (see Figures 2 and 3).

Stage 2-Rapid pressure drop When the material reaches a desired temperature near or below the phase transition temperature, the chamber is quickly or rapidly depressurized. This reduced pressure induces nucleation and phase transition of the solution in the vial or container. In a preferred embodiment, chamber depressurization is accomplished by opening or partially opening a large control valve that separates the high pressure chamber from the atmospheric environment, or a lower pressure chamber or environment. The increased pressure is quickly reduced by the mass flow of the gaseous atmosphere from the chamber. The reduced pressure needs to be fairly fast to induce nucleation. The decompression should be terminated in a few seconds or less, preferably 40 seconds or less, more preferably 20 seconds or less, and most preferably 10 seconds or less.

  In typical lyophilization applications, the pressure difference between the initial chamber pressure and the final chamber pressure after depressurization should be greater than about 7 psi. However, in some situations, a smaller pressure drop can induce nucleation. Most commercial freeze dryers can easily provide the pressure drop range required to control nucleation. Many freeze dryers are designed with pressure ratings in excess of 25 psig to withstand traditional sterilization procedures using saturated steam at 121 ° C. Such equipment ratings provide ample opportunity to induce nucleation according to a protocol that depressurizes from a starting pressure that exceeds atmospheric or ambient pressure. The increase in pressure and subsequent depressurization can be accomplished via any known means (eg, air, water, or mechanical). In a preferred embodiment, the operating pressure for the method should remain below the supercritical pressure of any applied gas, and during material nucleation, the material is at an extremely low pressure (ie, Exposure to about 10 mTorr or less) should be avoided.

  While not wishing to be bound by any particular mechanism, one possible mechanism for explaining the controlled nucleation observed in the practice of this method is the gas in solution in matter. Comes out of solution during decompression and forms bubbles, which nucleate the material. The initial elevated pressure increases the concentration of gas dissolved in the solution. The rapid pressure drop after cooling reduces the solubility of the gas and the subsequent release of the gas from the supercooled solution induces phase transition nucleation.

  Another possible mechanism is that during depressurization, the temperature drop of the gas proximate to the material results in a cold spot on the material surface, which initiates nucleation. Another possible mechanism is that the reduced pressure results in the evaporation of some liquid in the material, and the cooling that occurs from the endothermic evaporation process may initiate nucleation. Another possible mechanism is that the reduced cold gas closest to the material is either equilibrated with the material prior to depressurization or released from the material by evaporation during the vacuum. Freezing such vapor, the resulting solid particles reenter the material and act as seeds or surfaces to initiate nucleation. One or more of these mechanisms may contribute to the onset of freezing or solidification nucleation, to some extent, depending on the nature of the material, its environment and the phase transition being nucleated.

  This process may be performed exclusively at pressures that cover a pressure range that is greater than or spans the ambient pressure. For example, the initial chamber pressure may be higher than the ambient pressure, and after the final chamber pressure and decompression, it may be higher than the ambient pressure but lower than the initial chamber pressure. The initial chamber pressure may be higher than ambient pressure, and may be about the final chamber pressure, about ambient pressure after decompression, or slightly below ambient pressure.

  The rate and magnitude of the pressure drop is also believed to be an important aspect of the method. Experiments show that nucleation will be induced when the pressure drop (ΔP) is greater than about 7 psi. Alternatively, the magnitude of the pressure drop may be expressed as an absolute pressure ratio, R = Pi / Pf. Here, Pi is the initial absolute pressure, and Pf is the final absolute pressure. In many practical applications of the method, it is believed that nucleation will be induced upon depressurization when the absolute pressure ratio R is greater than about 1.2. The pressure drop rate also plays an important role in the method. One way to characterize the pressure drop rate is to use parameter A, where A = ΔP / Δt. Again, it is summarized that nucleation will be induced for a value of A greater than a predetermined value, such as about 0.2 psi / sec. Empirical data throughout the experiment must help to determine the preferred pressure drop and pressure drop rate.

  The following examples highlight various aspects and features of the method for inducing nucleation in matter disclosed herein and should not be taken in a limiting sense. Rather, these examples are for illustrative purposes only, and the scope of the present invention should be determined only by the claims appended hereto.

All examples described herein were performed in a pilot scale VirTis 51-SRC lyophilizer with four shelves totaling approximately 1.0 m ² and internal capacitors. The unit was modified to withstand positive pressures up to about 15 psig. A 1.5 ”diameter circular opening is also added to the rear wall of the freeze drying chamber, 1.5” diameter stainless steel extends from the hole, passes through the rear wall insulation, and exits from the back of the freeze dryer to the surface I attached a tube. The tube was fitted with two 1.5 "full flow, air-operated ball valves through a sanitary appliance. One ball valve allowed gas to flow into the lyophilization chamber, thereby up to 15 psig. The second ball valve causes the gas to flow out of the lyophilization chamber and reduce the chamber pressure to atmospheric conditions (0 psig) All refrigeration of the lyophilization shelf and condenser is made with Praxair NCool ( Performed via circulation of Dynalene MV heat transfer fluid cooled by liquid nitrogen using a trademark-HX system.

  All solutions were prepared in a class 100 clean room. The lyophilizer was installed with doors, shelves, and control equipment all accessible from the clean room, while other components (pumps, heaters, etc.) were placed in a non-clean room environment. All solutions were prepared with HPLC grade water (filtered through a 0.10 μm membrane, Fisher Scientific). The final solution was filtered through a 0.22 μm membrane prior to filling into vials or lyophilized containers. All gas was fed through a cylinder and filtered through a 0.22 μm filter to remove particles. Glass containers (5 mL vials and 60 mL bottles) were obtained from Wheaton Science Products with pre-washed particles. Where appropriate, pharmaceutically acceptable carriers were used. The above steps were taken to ensure materials and methods that meet conventional drug manufacturing standards for particles that act as nucleating agents.

  As used herein, “pharmaceutically acceptable carrier” refers to any and all solvents, dispersion media, antioxidants, salts, coatings, surfactants, preservatives (eg, methyl parahydroxybenzoate). Or propyl, sorbic acid, antibacterial agent, antifungal agent), isotonic agent, solution retarder (eg, paraffin), absorbent (eg, kaolin clay, bentonite clay), drug stabilizer (eg, sodium lauryl sulfate), Gels, binders (eg syrup, acacia, gelatin, sorbitol, tragacanth, polyvinylpyrrolidone, carboxymethylcellulose, alginate), excipients (eg lactose, lactose, polyethylene glycol), disintegrants (eg agar, starch, lactose) , Calcium phosphate, calcium carbonate, alginic acid, sorbitol, glycine , Spreading agents (eg cetyl alcohol, glycerol monostearate), lubricants, absorption enhancers (eg quaternary ammonium salts), edible oils (eg almond oil, coconut oil, oily esters or propylene glycol), sweeteners , Flavoring agents, coloring agents, fillers (eg, starch, lactose, sucrose, glucose, mannitol), tableting lubricants (eg, magnesium stearate, starch, glucose, lactose, rice flower, cucumber), inhalable carriers (eg, , Hydrocarbon high pressure gas), buffering agents, or the like and combinations thereof as would be known to one skilled in the art.

  For the experimental conditions described herein and all lyophilized formulations studied, stochastic nucleation is typically between about −8 ° C. and −20 ° C. and occasionally −5 ° C. It was observed to occur at a temperature of ° C. The container could generally be held at a temperature above -8 ° C for extended periods without nucleation. The onset of nucleation and subsequent crystal growth (ie, freezing) was determined by measuring the temperature as the point where the temperature of the vessel quickly increases in response to the exothermic latent heat of fusion. The start of freezing could also be determined visually through the viewing window in the freeze dryer chamber door.

Example 1-Control of Nucleation Temperature 2.5 mL of a 5 wt% mannitol solution was injected into four separate vials. The predicted thermodynamic freezing point of a 5 wt% mannitol solution is approximately -0.5 ° C. Four vials were placed close to each other and placed on a freeze dryer shelf. The temperature of the four vials was monitored using a thermocouple mounted on the surface. The lyophilizer was pressurized to 14 psig with argon.

  The freeze-dryer shelf was cooled and the vial temperature was between approximately -1.3 ° C and approximately -2.3 ° C (thermocouple measurement accuracy +/- 1 ° C). The lyophilizer was then depressurized from about 14 psig to about atmospheric pressure without taking 5 seconds to induce nucleation of the solution in the vial. All four vials nucleated immediately after depressurization and started to freeze. The results are summarized in Table 1 below.

  As seen in Table 1, the controlled nucleation temperature in this example (ie, the vial initial temperature) is very close to the predicted thermodynamic freezing point of the solution. In this way, the method makes it possible to control that nucleation occurs in solutions with a very low degree of supercooling, or at nucleation temperatures close to or just slightly cooler than their freezing point.

Example 2-Control of Nucleation Temperature In this example, 2.5 mL of a 5 wt% mannitol solution was injected into 95 vials. The thermodynamic freezing point of a 5 wt% mannitol solution is approximately -0.5 ° C. The 95 vials were placed close to each other and placed on the lyophilizer shelf. The temperature of six vials at different locations on the freeze dryer shelf was continuously monitored using a thermocouple mounted on the surface. The lyophilizer was pressurized to about 14 psig in an argon atmosphere. The shelf of the freeze dryer was then cooled to bring the vial temperature to around -5 ° C. The lyophilizer was then depressurized from about 14 psig to about atmospheric pressure without taking 5 seconds to induce nucleation of the solution in the vial. All 95 vials were visually observed to nucleate immediately after depressurization and to begin freezing. Thermocouple data for the six vials monitored monitored the visual observation. The results are summarized in Table 2.

  As can be seen, the controlled nucleation temperature (i.e., the initial temperature of the vial) in this example is somewhat lower than the expected thermodynamic freezing point of the solution. In this way, the method makes it possible to control the occurrence of nucleation in a moderately supercooled solution. This example also demonstrates the scalability of the method to a number of vial applications.

Example 3-Controlling the magnitude of vacuum In this example, 2.5 mL of a 5 wt% mannitol solution was injected into a plurality of vials. Again, the predicted thermodynamic freezing point of the 5 wt% mannitol solution is approximately -0.5 ° C. In each test run, the vials were placed on the freeze dryer shelf in close proximity to each other. Similar to the examples described above, the temperature of the vial was monitored using a thermocouple mounted on the surface. The argon atmosphere in the lyophilizer was pressurized to different pressures and the lyophilizer shelf was cooled so that the vial temperature was about -5 ° C. In each test run, the lyophilizer was then evacuated quickly (ie, without taking 5 seconds) from the selected pressure to atmospheric pressure, aiming to induce nucleation of the solution in the vial. . The results are summarized in Table 3.

  As seen in Table 3, controlled nuclei when the pressure drop is greater than about 7 psi and the nucleation temperature (ie, the vial initial temperature) is between about −4.7 ° C. and −5.8 ° C. Formation occurred.

Example 4-Depressurization Rate Control For this example, multiple vials were injected with 2.5 mL of a 5 wt% mannitol solution with a predicted thermodynamic freezing point of approximately -0.5 ° C. . In each test run with varying decompression times, the vials were placed on the freeze dryer shelf in close proximity to each other. Similar to the examples described above, the temperature of the vial was monitored using a thermocouple mounted on the surface. As in the above example, the argon atmosphere in the lyophilizer was pressurized to about 14 psig, the shelf was cooled, and the vial temperature was brought to approximately -5 ° C. In each test run, the lyophilizer was then evacuated at different evacuation rates from 14 psig to atmospheric pressure, aiming to induce nucleation of the solution in the vial.

  In order to examine the effect of the decompression speed or decompression time, a limiting ball valve was installed at the outlet of the decompression control valve on the back of the freeze dryer. When the restriction valve is fully opened, the decompression of about 14 psig to about 0 psig is completed in approximately 2.5 seconds. As long as the limiting valve is partially closed, the decompression time of the chamber can be lengthened. Using a restricted ball valve, several tests were performed with the freeze dryer chamber depressurized at different rates to determine or determine the effect of the depressurization rate on nucleation. The results are summarized in Table 4.

  As can be seen in Table 4, nucleation has a decompression time of less than 42 seconds, a pressure drop of greater than about 14 psi, and a nucleation temperature (ie, the initial vial temperature) of about −4.6 ° C. and about −5. Only happened if it was between 8 ° C. These results suggest that the decompression needs to be completed relatively quickly for this method to be effective.

Example 5-Controlling the Gas Atmosphere Approximately 2.5 mL of a 5 wt% mannitol solution was again injected into each of the plurality of vials and placed on the freeze dryer shelf in close proximity to each other. Similar to the examples described above, the temperature of the test vial was monitored using a thermocouple mounted on the surface. The gas atmosphere in the lyophilizer was changed while always maintaining a positive pressure of about 14 psig for different test operations. In this example, the freeze dryer shelf was cooled to bring the vial temperature from approximately -5 ° C to -7 ° C. In each test run, it was then aimed to rapidly depressurize the lyophilizer from about 14 psig to atmospheric pressure to induce nucleation of the solution in the vial. The results are summarized in Table 5.

  As can be seen, all except the helium gas atmosphere when the pressure drop is about 14 psi and the nucleation temperature (ie, the vial initial temperature) is between about -4.7 ° C and about -7.4 ° C. Controlled nucleation occurred in the gas atmosphere. Although not shown in the examples, it is believed that alternative conditions would allow controlled nucleation even in a helium atmosphere.

Example 6 Large Volume Solution In this example, about 30 mL of a 5 wt% mannitol solution with a predicted thermodynamic freezing point of approximately −0.5 ° C. is poured into six freeze-dried bottles (60 mL capacity). did. Six freeze-drying bottles were placed on the freeze-dryer shelf in close proximity to each other. The temperature of six bottles placed at different locations on the freeze dryer shelf was monitored with a surface mounted thermocouple. The lyophilizer was pressurized to about 14 psig in an argon atmosphere. The shelf of the freeze dryer was then cooled to bring the bottle temperature to around -5 ° C. The lyophilizer was then depressurized from 14 psig to about atmospheric pressure without taking 5 seconds to induce nucleation of the solution in the bottle. The results are summarized in Table 6.

  In another experiment, approximately 1000 mL of a 5 wt% mannitol solution was placed in a large plastic lyophilization tray (capacity 1800 mL, Gore LYOGUARD). The resulting tray was pre-cleaned to meet the US Pharmacopoeia low particle requirements. The tray was placed on a lyophilizer shelf and the temperature of the tray was monitored by a thermocouple mounted on the outer surface of the tray near the center on one side. The shelf of the freeze dryer was then cooled to bring the tray temperature to around -7 ° C. The lyophilizer was then depressurized from 14 psig to about atmospheric pressure without taking 5 seconds to induce nucleation of the solution in the tray. The results are also summarized in Table 6.

  As in the above example, all containers nucleated immediately after decompression and began to freeze. Also, as in the above example, the nucleation temperature (i.e., vessel temperature) in this example was very well controllable so that it was slightly closer to the thermodynamic freezing temperature of the solution. More importantly, this example illustrates that the method allows control of the occurrence of nucleation in larger volumes of solution and various shaped containers. It should be noted that the efficiency of the vacuum process would be expected to improve with increasing formulation volume. This is because nucleation events are more likely to occur when more molecules are present and aggregate and form critical nuclei.

Example 7 Dynamic Cooling vs. Equilibrium Cooling The present nucleation control method can be used in various forms. Examples 1-6 above each control the nucleation temperature of a lyophilized solution that is essentially equilibrated at a temperature below the thermodynamic freezing point (ie, a very slowly changing temperature). This aspect is demonstrated. This example demonstrates that nucleation can occur even at temperatures below the thermodynamic freezing point in a dynamic cooling environment (ie, the solution undergoing a rapid temperature change).

  In this example, vials 1-6 represent the samples described above in connection with Example 2. In addition, three separate vials (vials 7-9) were also injected with 2.5 mL of a 5 wt% mannitol solution. In another test operation, three additional vials were placed on the freeze dryer shelf in close proximity to each other. The lyophilizer shelf was rapidly cooled to a final shelf temperature of -45 ° C. When one of the vials reached a temperature of about −5 ° C. as measured by a surface mount thermocouple, the freeze dryer was rapidly depressurized from about 14 psig to 0 psig to aim for nucleation induction. All three vials nucleated immediately after depressurization and began to freeze. As a result of the dynamic cooling environment, the temperature of the vial dropped significantly between −6.8 ° C. and −9.9 ° C. prior to nucleation. The comparison results are summarized in Table 7 below.

  The effectiveness of this method of controlling nucleation in a lyophilized solution equilibrated at a given temperature range, or a lyophilized solution that is dynamically cooled, allows the end user to use it with different advantages and tradeoffs. Provides two possible modalities. By equilibrating the lyophilized solution, the nucleation temperature range will be narrowed or minimized to the performance limits of the lyophilizer itself. The equilibration step may require extra time to achieve compared to conventional or dynamic freezing procedures where the chamber and vial temperatures are lowered below about −40 ° C. in one step. Absent. However, using an equilibration step yields other benefits associated with precise control of the nucleation temperature of the material, along with much improved nucleation uniformity across all vials or containers. It must be.

  Alternatively, if it is not desired to equilibrate the temperature of the material or lyophilized solution, a vacuum step may simply be performed at an appropriate time during normal freezing or dynamic cooling procedures. Depressurization during dynamic cooling will broaden the nucleation temperature for the material in the lyophilization vessel, but minimizes the time added to the freezing procedure and alleviates the problem of extreme supercooling Will.

Example 8-Effect of different excipients This method of controlling or inducing nucleation in a material can be used to control the nucleation temperature of a supercooled solution containing different lyophilized excipients. This example demonstrates the use of this method with the following excipients: mannitol, hydroxyethyl starch (HES), polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), dextran, glycine, sorbitol, sucrose, and Trehalose. For each excipient, two vials were injected with 2.5 mL of a solution containing 5 wt% excipient. These vials were placed on the freeze dryer shelf in close proximity to each other. The lyophilizer was pressurized to about 14 psig in an argon atmosphere. The lyophilizer shelf was cooled to bring the vial temperature to around -3 ° C and then rapidly depressurized to induce nucleation. The results are summarized in Table 8.

Example 9-Control of Nucleation of Protein Solution The method disclosed herein can be used to control the nucleation temperature of a supercooled protein solution without negative or adverse effects on protein solubility or enzyme activity. Can be used. In this example, two proteins, bovine serum albumin (BSA) and lactate dehydrogenase (LDH) were used.

  BSA was dissolved in 5 wt% mannitol at a concentration of 10 mg / mL. Three freeze-dried vials were injected with 2.5 mL of BSA-mannitol solution, placed close to each other and placed on the freeze-dryer shelf. The lyophilizer was pressurized to about 14 psig in an argon atmosphere. The shelf of the freeze dryer was cooled and the temperature of the vial was brought to around -5 ° C. The lyophilizer was rapidly depressurized to induce nucleation. All vials of BSA solution nucleated immediately after depressurization and began to freeze. No protein precipitation was observed upon thawing.

  The LDH protein was obtained from two different suppliers and was named LDH-1 or LDH-2 to distinguish the two different batches for purposes of clarity. LDH-1 was dissolved in 5 wt% mannitol at a concentration of 1 mg / mL. Six freeze-dried vials were injected with 2.5 mL of LDH-1 / mannitol solution, placed close to each other and placed on the freeze-dryer shelf. The lyophilizer was pressurized to about 14 psig in an argon atmosphere. The lyophilizer shelf was cooled starting at room temperature until the vial temperature was around -4 ° C. The lyophilizer was then rapidly depressurized to induce nucleation. All vials nucleated immediately after decompression and began to freeze. The vial was held in this state for about 15 minutes. The freeze-dryer shelf was then cooled at a rate of approximately 1 ° C./min, the vial temperature was near −45 ° C. and held for an additional 15 minutes to ensure completion of the freezing process. After the freezing phase, the lyophilizer shelf was then warmed at a rate of about 1 ° C./min, and the vial temperature was raised to around 5 ° C. No protein precipitation was observed upon thawing. The contents of the vial were assayed for enzyme activity and these results were compared to a control sample of unfrozen LDH-1 / mannitol solution.

  As part of Example 9, a sample of LDH-1 / mannitol solution that had been nucleated under reduced pressure was compared to a sample that stochastically nucleated. In probabilistic nucleated LDH-1 samples, the freezing procedure was repeated without pressurization and depressurization, and without an argon atmosphere. Specifically, LDH-1 was dissolved in 5 wt% mannitol at a concentration of 1 mg / mL. Six freeze-dried vials were injected with 2.5 mL of LDH-1 / mannitol solution and placed on the freeze-dryer shelf in close proximity to each other. The lyophilizer shelf was cooled at a rate of about 1 ° C./min starting from room temperature and the vial temperature was held near −45 ° C. for 15 minutes to ensure the freezing process was completed. After the freeze phase, the freeze dryer shelf was warmed at a rate of about 1 ° C./min and the vial temperature was raised to around 5 ° C. During thawing, no protein precipitation was observed. The contents of the vial were assayed for enzyme activity and the results were compared to the same control sample of unfrozen LDH-1 / mannitol solution. Again as part of Example 9, the above experiment on LDH-1 was repeated using LDH-2. The only difference was that the nucleation temperature was not around -3 ° C for LDH-2, but -4 ° C for LDH-1.

  As can be seen in Table 9, the controlled nucleation and freezing process achieved via reduced pressure does not clearly reduce enzyme activity over the comparative stochastic nucleation and freezing procedure. . In fact, the controlled nucleation process achieved through reduced pressure has a mean activity loss of only 17.8% for LDH-1 and only 26.5% for LDH-2, which is stochastic. After complete nucleation, the average activity loss seems to maintain better enzyme activity compared to 35.9% for LDH-1 and 41.3% for LDH-2 It is.

  It should be noted that the stochastic nucleation temperature observed for LDH-2 was substantially higher than the stochastic nucleation temperature for LDH-1. This difference may be due to any contaminants in LDH-2 that act as nucleating agents. Compared to LDH-1, LDH-2 has a stochastic nucleation temperature much closer to the controlled nucleation temperature, but nevertheless for LDH-1 and LDH-2 controlled nucleation The improvement in retention of enzyme activity obtained through is similar to 18.1% and 14.8%, respectively. This result suggests that improvements in retention of enzyme activity can be attributed, in part, to the characteristics of the controlled nucleation process itself, as well as the predetermined higher nucleation temperature obtained through reduced pressure. ing.

Example 10-Reduction of Primary Drying Time About 10.01 g of mannitol was mixed with about 190.07 g of water to prepare a 5 wt% mannitol solution. A vial was injected with 2.5 mL of a 5 wt% mannitol solution. Empty and filled vials were weighed to determine the mass of water added to the vial. Twenty vials were placed close together and placed in a rack on the lyophilizer shelf. The temperature of the six vials was monitored using a surface mount thermocouple. All monitored vials were surrounded by other vials to improve the uniformity of vial behavior. The lyophilizer was pressurized to about 14 psig in a controlled gaseous atmosphere of argon gas. The lyophilizer shelf was cooled from room temperature to about −6 ° C., and the vial temperature was between approximately −1 ° C. and −2 ° C. The lyophilizer was then depressurized from about 14 psig to about atmospheric pressure without taking 5 seconds to induce nucleation of the solution in the vial. All vials visually observed or monitored with thermocouples nucleated immediately after depressurization and began to freeze.

  The shelf temperature was then rapidly lowered to about −45 ° C. to complete the freezing process. Once the temperature of all vials was below about −40 ° C., the lyophilization chamber was evacuated and the primary drying process (ie, sublimation) was initiated. During this drying process, the freeze dryer shelf was warmed to about -14 ° C over 1 hour and held at that temperature for 16 hours. The condenser was maintained at about -60 ° C throughout the drying process. The vacuum pump was turned off to stop primary drying, and argon was returned to the chamber to atmospheric pressure. The vial was immediately removed from the lyophilizer and weighed to determine how much water was lost during the primary drying process.

  In another experiment as part of Example 10, another vial was injected with 2.5 mL of the same 5 wt% mannitol solution. Empty and filled vials were weighed to determine the mass of water added to the vial. The vials were placed in a lyophilizer in the same manner as above, and the temperature of the six vials was again monitored using a surface mount thermocouple. The lyophilizer shelf was rapidly cooled from room temperature to about −45 ° C. to freeze the vials. Nucleation occurred stochastically between about −15 ° C. and about −18 ° C. during the cooling phase. Once all the vials were below about −40 ° C., the vials were dried in the same manner as described above. At the end of primary drying, the sample was immediately removed from the freeze dryer and weighed to determine how much water was lost during the primary drying process.

  The results of the lyophilization process with controlled nucleation and stochastic nucleation are summarized in Table 10. It should be noted that these two experiments differ only in the addition of a controlled nucleation through a decompression step to one experiment. As can be seen in Table 10, the controlled nucleation process achieved through vacuum is at a very low degree of subcooling, in this example between about −1.1 ° C. and −2.3 ° C. Nucleated. A much higher nucleation temperature for the controlled nucleation case as compared to the stochastic nucleation case yields ice structures and resulting frozen cakes with dramatically improved drying performance . For the same length of drying time, vials nucleated using the disclosed vacuum method between about -1.1 ° C and -2.3 ° C averaged their water Vials that lost 86.1%, but stochastically nucleated between about -14.5 ° C and -17.9 ° C lost only 65.3% on average. There wasn't. Thus, in order to cause a stoichiometrically nucleated vial to lose as much water as a nucleated vial in a controlled manner according to the method disclosed herein, a much longer primary drying Will take time. The improvement in drying time is likely to be attributed to the formation of larger ice crystals at higher nucleation temperatures. These larger ice crystals leave larger pores during sublimation, and these larger pores have less resistance to water vapor flow during further sublimation.

  The method provides an improved method of controlling the temperature and / or time at which the supercooled material, ie, liquid or solution, nucleates and then freezes. Although this application focuses in part on lyophilization, a similar problem arises for any material processing step involving nucleated phase transitions. Examples of such treatments are polymer and metal crystallization from melts, material crystallization from supersaturated solutions, protein crystallization, artificial snow production, food freezing, freeze concentration, fractional crystallization, low temperature Includes storage, or condensation of the vapor into a liquid.

  The most direct benefit of controlling the nucleation temperature of a liquid or solution is the ability to control the number and size of solid domains produced by the phase transition. For example, in water that is freezing, the nucleation temperature directly controls the size and number of ice crystals formed. Generally speaking, when the nucleation temperature is higher, ice crystals are smaller in number and larger in size.

  The ability to control the number and size of solid domains produced by phase transitions may provide additional benefits. For example, in the lyophilization process, the number and size of ice crystals strongly affects the drying characteristics of the lyophilized cake. Larger ice crystals produced by higher nucleation temperatures leave larger pores during sublimation and the larger pores provide less resistance to water vapor flow during subsequent sublimation. Thus, the present method provides a means to increase the primary drying (ie sublimation) rate during the lyophilization process by increasing the nucleation temperature.

  Another potential benefit may be realized in applications that store sensitive materials (ie, cryopreservation) via a freezing process. For example, but not limited to, mammalian tissue samples (eg, umbilical cord blood, tissue biopsy, egg cells and sperm cells), cell lines (eg, mammalian, yeast, prokaryotic cells) frozen in aqueous solution Biological materials, including biological, fungal, and other cell lines) and biological molecules (eg, proteins, DNA, RNA and their subnets) may experience various stresses during the freezing process. This can impair the function or activity of the substance. Ice formation can physically disrupt the material or create drastic changes in the interfacial bonding, osmotic pressure, solute concentration, etc. experienced by the material. Since nucleation controls the structure and dynamics of ice formation, it can significantly affect these stresses. Thus, the disclosed method provides a unique means of relieving stress associated with the cryopreservation process and improving the recovery of function or activity from cryopreserved materials. This method is in contrast to traditional nucleation control methods {eg, sowing or contacting with cold surfaces) used to initiate extracellular ice formation in a two-step cryopreservation algorithm designed for living cells. Even shows an improvement.

  The method is also applicable to complex solutions or mixtures containing several components in both cryopreservation and lyophilization applications. These formulations are pharmaceutically active ingredients (eg, synthetic reagents, proteins, peptides, or vaccines) with aqueous, organic, or water-organic mixed solvents, and optionally the physical properties of the active ingredient during drying. Bulking agents (eg, dextrose, glucose, glycine, lactose, maltose, mannitol, polyvinylpyrrolidone, sodium chloride, and sorbitol) that help prevent chemical loss; buffering agents that help maintain an appropriate environmental pH or toxicity for the active ingredient Toxicity modifiers (eg acetic acid, benzoic acid, citric acid, hydrochloric acid, lactic acid, maleic acid, phosphoric acid, tartaric acid, and sodium salts of the aforementioned acids); active during processing or in the final liquid or dry form Stabilizers that help retain the structure or function of the ingredients (eg, alanine, dimethyl sulfoxide, Rolls, glycine, human serum albumin, polyethylene glycol, lysine, polysorbate, sorbitol, sucrose, and trehalose); agents that modify the glass transition behavior of the formulation (eg, polyethylene glycol and sugar), and protect the active ingredient from degradation It may be a solution containing one or more relaxation ingredients including antioxidants (eg, ascorbate, sodium bisulfite, sodium formaldehyde, sodium metabisulfite, sodium sulfite, sulfoxylate, and thioglycerol) Many.

  Since nucleation is typically a random process, multiple identical materials exposed to the same processing conditions may nucleate at different temperatures. As a result, the properties of these materials that depend on the nucleation behavior will probably be different, despite the same processing conditions. The disclosed method provides a way to control the nucleation temperature of multiple substances simultaneously, thereby presenting a way to increase the uniformity of their product properties that depend on the nucleation behavior. In a typical lyophilization process, for example, the same solution in separate vials can probabilistically nucleate over a wide range of temperatures and, as a result, the final, lyophilized Products may have significant variability in important properties such as residual moisture, activity and time to return to liquid. By controlling the nucleation temperature through the process disclosed herein, the bottle-to-bottle uniformity of the product characteristics resulting from the lyophilization process can be dramatically improved.

  The ability to control a material's nucleation behavior may also provide substantial benefits in reducing the time required to develop an industrial process that depends on normal uncontrolled nucleation events. is there. For example, successful lyophilization that can be achieved in a reasonable amount of time, produces desired product properties within a specified homogeneity range, and maintains sufficient activity of an active pharmaceutical ingredient (API). It often takes months to develop a cycle. By providing a means of controlling nucleation and thereby potentially improving primary drying time, product homogeneity, and API activity, the method develops a successful dry freezing procedure. The time required for this must be drastically reduced.

  In particular, the potential benefits of the nucleation process provide increased flexibility in identifying the composition of the formulation to be lyophilized. Since controlled nucleation can better retain the API during the freezing phase, users can minimize the addition of relaxation components (eg, stabilizers) to the formulation, or more simply It must be possible to select a combination of prescription components to achieve the combined stability and processing objectives. Synergistic if controlled nucleation inherently increases the primary drying time (eg, by reducing the glass transition temperature of the aqueous solution) and minimizes the use of stabilizers or other relaxation components. Profits can be generated.

  The disclosed method is particularly well suited for large scale production or manufacturing operations. This is because it can be performed using the same equipment and process parameters that can be easily augmented or adapted to the production of a wide range of products. This process involves the process where all operations can be performed in a single chamber (eg lyophilizer) and the use of vacuum, additives, vibration, electric freezing etc. to induce nucleation. Take steps to nucleate matter using methods that do not require it.

  In contrast to the prior art, the method does not add anything to the lyophilized product. The method only requires that the material (eg, liquid in a vial) be initially held at a specific pressure under a gaseous atmosphere and that the pressure be rapidly reduced to a lower pressure. Any applied gas will be removed from the vial during the lyophilization cycle. The vials or their contents do not touch or touch anything except gas. Simple manipulation of ambient pressure and gaseous environment by itself is sufficient to achieve that goal. By inducing nucleation relying solely on changes in ambient pressure, the presently disclosed method affects all vials in the lyophilizer uniformly and simultaneously.

  This embodiment is also cheaper and easier to implement and maintain than conventional methods that affect nucleation in materials in lyophilization applications. The method allows for significantly faster primary drying during the lyophilization process, thereby reducing processing costs for the lyophilized drug. The method produces a lyophilized product that is much more uniform than conventional methods, thereby reducing product loss and barriers to entry of processors that cannot meet the more stringent uniformity specifications. Create. This method wins these benefits without contaminating the lyophilized product. Greater control of the process must result in improved output and reduced process time.

  From the foregoing, it should be appreciated that the present invention thus provides a method for inducing nucleation in a material and / or a method for freezing a material. Various modifications, changes, and variations of the method will be apparent to those skilled in the art, and such modifications, changes, and variations are within the scope of this application and the spirit and scope of the claims. It should be understood that it should be included.

FIG. 5 is a graph depicting temperature versus time for a solution undergoing a probabilistic nucleation process and further illustrating the range of nucleation temperatures for the solution. FIG. 5 is a graph depicting temperature versus time for a solution undergoing an equilibration cooling process with reduced nucleation consistent with the present method. FIG. 4 is a graph depicting temperature versus time for a solution undergoing a dynamic cooling process with reduced pressure nucleation consistent with the present method.

Claims (33)

Bringing the substance to a temperature near or below the phase transition temperature; and
Reducing the immediate pressure of the material to induce nucleation of phase transitions in the material;
A method of inducing nucleation of a phase transition in a substance containing   The method of claim 1, wherein the material is cooled to a metastable state.   The method of claim 1, further comprising continuing to cool the nucleated material after depressurization to a final or lower temperature to ensure that the phase transition of the material is complete.   2. A method according to claim 1 wherein depressurization is initiated when the material reaches a desired nucleation temperature.   The method of claim 1, wherein the depressurization is initiated for a desired time after the start of the cooling phase and when the temperature of the material is below the phase transition temperature.   The method of claim 1, further comprising returning the material to a liquid state.   The method of claim 1, wherein the substance is selected from the group consisting of a gas, a liquid, a solution, a suspension, a mixture, or a component of a suspension, solution, or mixture.   The method of claim 1, wherein the material is a solution and the phase transition temperature is a thermodynamic freezing point of the solution.   2. The material of claim 1, wherein the substance is a solution with one or more dissolved materials, and the phase transition temperature is a saturation temperature at which the dissolved materials will precipitate or crystallize from the solution. Method.   The method of claim 7, wherein the substance further comprises a biopharmaceutical substance, a drug substance, a chemical substance, a biological substance, a foodstuff, or a combination thereof.   The method of claim 1, wherein the substance is confined in the presence of a gaseous atmosphere.   The method of claim 11, wherein the gaseous atmosphere comprises argon, nitrogen, helium, air, water vapor, oxygen, carbon dioxide, neon, xenon, krypton, hydrogen, or mixtures thereof.   The method of claim 11, further comprising pressurizing the atmosphere surrounding the material.   The method of claim 1, wherein prior to depressurization, the material is first cooled from the phase transition temperature to a temperature in the range of 20 ° C. below the phase transition temperature.   The method of claim 1, wherein prior to depressurization, the material is cooled to a temperature in the range of about 5 ° C. below the phase transition temperature from the phase transition temperature.   The method of claim 1, wherein the pressure is reduced by an amount greater than about 14 psi.   The method of claim 1, wherein the pressure is reduced by an amount greater than about 7 psi.   The method of claim 1, wherein the pressure is lowered such that the absolute pressure ratio, Pi / Pf, is about 1.2 or greater.   The method of claim 1, wherein the pressure is reduced at a pressure drop rate greater than about 0.2 psi per second, ΔP / Δt.   The method of claim 1, wherein the pressure is reduced in 40 seconds or less.   7. The method of claim 6, wherein the substance comprises a component comprising a live or attenuated virus; a nucleic acid; a monoclonal or polyclonal antibody; a biomolecule; a non-peptide analog; a peptide;   The method of claim 21, wherein the component of the material returned to the liquid state exhibits a function or activity similar to the function or activity associated with the component of the starting material.   The method of claim 21, wherein the component of the material returned to a liquid state exhibits an improved function or activity relative to the function or activity associated with the stochastically nucleated material component.   The method of claim 21, wherein the component of the material returned to the liquid state exhibits a structure similar to the structure associated with the component of the starting material.   The method of claim 21, wherein the substance is retained in a plurality of containers, and the returned liquid substance from all containers exhibits uniform properties. Cooling the substance at a predetermined cooling rate;
Nucleating the material by rapidly reducing the pressure;
Continuing to cool the nucleated material to a predetermined final temperature to completely freeze the material;
For controlling the freezing process of a substance containing.   27. The method of claim 26, wherein depressurization is initiated when the material reaches a desired nucleation temperature.   27. The method of claim 26, wherein depressurization is initiated at a desired time after the beginning of the cooling phase and when the temperature of the material is below the phase transition temperature. Cooling the material to a temperature near or below the phase transition temperature;
Reducing the pressure in the immediate vicinity of the material to induce nucleation of the material;
Continuing cooling of the nucleated material to a predetermined final temperature to promote solidification of the material;
Solidification method including.   The substance is a solution having one or more dissolved materials, and the step of reducing the pressure closest to the substance reduces the pressure closest to the solution to reduce the pressure of the one or more materials in the solution; 30. The solidification method of claim 29, further comprising inducing phase transition nucleation. Cooling the gas to a temperature near or below the phase transition temperature;
Reducing the pressure to induce nucleation of phase transitions in the gas;
Continuing the cooling of the nucleated gas to a predetermined final temperature to condense the gas;
Control method for condensing gas including Warming the material to a temperature near or above the phase transition temperature;
Inducing a phase transition of the substance by lowering the immediate pressure on the substance;
A method for inducing a phase transition in a substance containing Bringing the substance to a temperature near the phase transition temperature in a pressurized gas atmosphere;
Reducing the pressure to induce a phase transition of the substance;
A phase transition method for a substance containing

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